Novel glucosaminidase

ABSTRACT

The invention provides glucosaminidase polypeptides and DNA (RNA) encoding glucosaminidase polypeptides and methods for producing such polypeptides by recombinant techniques. Also provided are methods for utilizing glucosaminidase polypeptides to screen for antibacterial compounds.

RELATED APPLICATIONS

[0001] This application is a Continuation-in-Part of U.S. patentapplication Ser. No. 08/899,088 filed, Jul. 23, 1997 which claimedbenefit of US Provisional Patent Application No. 60/011,888, filed Feb.20, 1996, PCT Application, International Application No. PCT/US97/02547,filed Feb. 19, 1997, and PCT Application, International Application No.PCT/US97/02318, filed Feb. 19, 1997.

FIELD OF THE INVENTION

[0002] This invention relates to newly identified polynucleotides andpolypeptides, and their production and uses, as well as their variants,agonists and antagonists, and their uses. In particular, in these and inother regards, the invention relates to novel polynucleotides andpolypeptides of the autolysin family, hereinafter referred to as“glucosaminidase”.

BACKGROUND OF THE INVENTION

[0003] It is particularly preferred to employ Staphylococcal genes andgene products as targets for the development of antibiotics. TheStaphylococci make up a medically important genera of microbes. They areknown to produce two types of disease, invasive and toxigenic. Invasiveinfections are characterized generally by abscesss formation effectingboth skin surfaces and deep tissues. Staphylococcus aureus is the secondleading cause of bacteremia in cancer patients. Osteomyelitis, septicarthritis, septic thrombophlebitis and acute bacterial endocarditis arealso relatively common. There are at least three clinical conditionsresulting from the toxigenic properties of Staphylococci. Themanifestation of these diseases result from the actions of exotoxins asopposed to tissue invasion and bacteremia. These conditions include:Staphylococcal food poisoning, scalded skin syndrome and toxic shocksyndrome

[0004] The frequency of Staphylococcus aureus infections has risendramatically in the past 20 years. This has been attributed to theemergence of multiply antibiotic resistant strains and an increasingpopulation of people with weakened immune systems. It is no longeruncommon to isolate Staphylococcus aureus strains which are resistant tosome or all of the standard antibiotics. This has created a demand forboth new anti-microbial agents and diagnostic tests for this organism.

[0005] Autolysin enzymes are able to disrupt the peptidoglycan chains ofbacterial cell walls. It is thought they are employed by bacteria in acarefully regulated manner in essential cell processes such as cell wallgrowth and septation. Inhibition of their action or disruption of theircontrolled function represents a probable antibacterial target.

[0006] Clearly, there is a need for factors, such as the novel compoundsof the invention, that have a present benefit of being useful to screencompounds for antibiotic activity. Such factors are also useful todetermine their role in pathogenesis of infection, dysfunction anddisease. There is also a need for identification and characterization ofsuch factors and their antagonists and agonists which can play a role inpreventing, ameliorating or correcting infections, dysfunctions ordiseases.

[0007] The polypeptides of the invention have amino acid sequencehomology to a known sp|P52081|ATL_STAAU AUTOLYSIN PRECURSOR protein(autolysin AtlE; GENBANK: locus SEU71377, accession U71377).

SUMMARY OF THE INVENTION

[0008] It is an object of the invention to provide polypeptides thathave been identified as novel glucosaminidase polypeptides by homologybetween the amino acid sequence set out in Table 1 [SEQ ID NO: 2] and aknown amino acid sequence or sequences of other proteins such assp|P52081|ATL_STAAU AUTOLYSIN PRECURSOR protein.

[0009] It is a further object of the invention to providepolynucleotides that encode glucosaminidase polypeptides, particularlypolynucleotides that encode the polypeptide herein designatedglucosaminidase.

[0010] In a particularly preferred embodiment of the invention, thepolynucleotide comprises a region encoding glucosaminidase polypeptidescomprising the sequence set out in Table 1 [SEQ ID NO:1] which includesa full length gene, or a variant thereof.

[0011] In another particularly preferred embodiment of the invention,there is a novel glucosaminidase protein from Staphylococcus aureuscomprising the amino acid sequence of Table 1 [SEQ ID NO:2], or avariant thereof.

[0012] In accordance with another aspect of the invention, there isprovided an isolated nucleic acid molecule encoding a mature polypeptideexpressible by the Staphylococcus aureus WCUH 29 strain contained in thedeposited strain.

[0013] As a further aspect of the invention, there are provided isolatednucleic acid molecules encoding glucosaminidase, particularlyStaphylococcus aureus glucosaminidase, including mRNAs, cDNAs, genomicDNAs. Further embodiments of the invention include biologically,diagnostically, prophylactically, clinically or therapeutically usefulvariants thereof, and compositions comprising the same.

[0014] In accordance with another aspect of the invention, there isprovided the use of a polynucleotide of the invention for therapeutic orprophylactic purposes, in particular genetic immunization. Among theparticularly preferred embodiments of the invention are naturallyoccurring allelic variants of glucosaminidase and polypeptides encodedthereby.

[0015] As another aspect of the invention, there are provided novelpolypeptides of Staphylococcus aureus referred to herein asglucosaminidase as well as biologically, diagnostically,prophylactically, clinically or therapeutically useful variants thereof,and compositions comprising the same.

[0016] Among the particularly preferred embodiments of the invention arevariants of glucosaminidase polypeptide encoded by naturally occurringalleles of the glucosaminidase gene.

[0017] In a preferred embodiment of the invention, there are providedmethods for producing the aforementioned glucosaminidase polypeptides.

[0018] In accordance with yet another aspect of the invention, there areprovided inhibitors to such polypeptides, useful as antibacterialagents, including, for example, antibodies.

[0019] In accordance with certain preferred embodiments of theinvention, there are provided products, compositions and methods forassessing glucosaminidase expression, treating disease, for example,disease, such as, infections of the upper respiratory tract (e.g.,otitis media, bacterial tracheitis, acute epiglottitis, thyroiditis),lower respiratory (e.g., empyema, lung abscesss), cardiac (e.g.,infective endocarditis), gastrointestinal (e.g., secretory diarrhoea,splenic abscess, retroperitoneal abscess), CNS (e.g., cerebral abscess),eye (e.g., blepharitis, conjunctivitis, keratitis, endophthalmitis,preseptal and orbital cellulitis, darcryocystitis), kidney and urinarytract (e.g., epididymitis, intrarenal and perinephric abscess, toxicshock syndrome), skin (e.g., impetigo, folliculitis, cutaneousabscesses, cellulitis, wound infection, bacterial myositis) bone andjoint (e.g., septic arthritis, osteomyelitis), assaying geneticvariation, and administering a glucosaminidase polypeptide orpolynucleotide to an organism to raise an immunological response againsta bacteria, especially a Staphylococcus aureus bacteria.

[0020] In accordance with certain preferred embodiments of this andother aspects of the invention, there are provided polynucleotides thathybridize to glucosaminidase polynucleotide sequences, particularlyunder stringent conditions.

[0021] In certain preferred embodiments of the invention, there areprovided antibodies against glucosaminidase polypeptides.

[0022] In other embodiments of the invention, there are provided methodsfor identifying compounds which bind to or otherwise interact with andinhibit or activate an activity of a polypeptide or polynucleotide ofthe invention comprising: contacting a polypeptide or polynucleotide ofthe invention with a compound to be screened under conditions to permitbinding to or other interaction between the compound and the polypeptideor polynucleotide to assess the binding to or other interaction with thecompound, such binding or interaction being associated with a secondcomponent capable of providing a detectable signal in response to thebinding or interaction of the polypeptide or polynucleotide with thecompound; and determining whether the compound binds to or otherwiseinteracts with and activates or inhibits an activity of the polypeptideor polynucleotide by detecting the presence or absence of a signalgenerated from the binding or interaction of the compound with thepolypeptide or polynucleotide.

[0023] In accordance with yet another aspect of the invention, there areprovided glucosaminidase agonists and antagonists, preferablybacteriostatic or bacteriocidal agonists and antagonists.

[0024] In a further aspect of the invention, there are providedcompositions comprising a glucosaminidase polynucleotide or aglucosaminidase polypeptide for administration to a cell or to amulticellular organism.

[0025] Various changes and modifications within the spirit and scope ofthe disclosed invention will become readily apparent to those skilled inthe art from reading the following descriptions and from reading theother parts of the present disclosure.

GLOSSARY

[0026] The following definitions are provided to facilitateunderstanding of certain terms used frequently herein.

[0027] “Host cell” is a cell which has been transformed or transfected,or is capable of transformation or transfection by an exogenouspolynucleotide sequence.

[0028] “Identity,” as known in the art, is a relationship between two ormore polypeptide sequences or two or more polynucleotide sequences, asdetermined by comparing the sequences. In the art, “identity” also meansthe degree of sequence relatedness between polypeptide or polynucleotidesequences, as the case may be, as determined by the match betweenstrings of such sequences. “Identity” and “similarity” can be readilycalculated by known methods, including but not limited to thosedescribed in (Computational Molecular Biology, Lesk, A. M., ed., OxfordUniversity Press, New York, 1988; Biocomputing: Informatics and GenomeProjects, Smith, D. W., ed., Academic Press, New York, 1993; ComputerAnalysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G.,eds., Humana Press, New Jersey, 1994; Sequence Analysis in MolecularBiology, von Heinje, G., Academic Press, 1987; and Sequence AnalysisPrimer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York,1991; and Carillo, H., and Lipman, D., SIAM J. Applied Math., 48: 1073(1988). Preferred methods to determine identity are designed to give thelargest match between the sequences tested. Methods to determineidentity and similarity are codified in publicly available computerprograms. Preferred computer program methods to determine identity andsimilarity between two sequences include, but are not limited to, theGCG program package (Devereux, J., et al., Nucleic Acids Research 12(1):387 (1984)), BLASTP, BLASTN, and FASTA (Atschul, S. F. et al., J. Molec.Biol. 215: 403-410 (1990). The BLAST X program is publicly availablefrom NCBI and other sources (BLAST Manual, Altschul, S., et al., NCBINLM NIH Bethesda, Md. 20894; Altschul, S., et al., J. Mol. Biol. 215:403-410 (1990). As an illustration, by a polynucleotide having anucleotide sequence having at least, for example, 95% “identity” to areference nucleotide sequence of SEQ ID NO: 1 it is intended that thenucleotide sequence of the polynucleotide is identical to the referencesequence except that the polynucleotide sequence may include up to fivepoint mutations per each 100 nucleotides of the reference nucleotidesequence of SEQ ID NO: 1. In other words, to obtain a polynucleotidehaving a nucleotide sequence at least 95% identical to a referencenucleotide sequence, up to 5% of the nucleotides in the referencesequence may be deleted or substituted with another nucleotide, or anumber of nucleotides up to 5% of the total nucleotides in the referencesequence may be inserted into the reference sequence. These mutations ofthe reference sequence may occur at the 5 or 3 terminal positions of thereference nucleotide sequence or anywhere between those terminalpositions, interspersed either individually among nucleotides in thereference sequence or in one or more contiguous groups within thereference sequence. Analogously, by a polypeptide having an amino acidsequence having at least, for example, 95% identity to a reference aminoacid sequence of SEQ ID NO:2 is intended that the amino acid sequence ofthe polypeptide is identical to the reference sequence except that thepolypeptide sequence may include up to five amino acid alterations pereach 100 amino acids of the reference amino acid of SEQ ID NO: 2. Inother words, to obtain a polypeptide having an amino acid sequence atleast 95% identical to a reference amino acid sequence, up to 5% of theamino acid residues in the reference sequence may be deleted orsubstituted with another amino acid, or a number of amino acids up to 5%of the total amino acid residues in the reference sequence may beinserted into the reference sequence. These alterations of the referencesequence may occur at the amino or carboxy terminal positions of thereference amino acid sequence or anywhere between those terminalpositions, interspersed either individually among residues in thereference sequence or in one or more contiguous groups within thereference sequence.

[0029] “Isolated” means altered “by the hand of man” from its naturalstate, i.e., if it occurs in nature, it has been changed or removed fromits original environment, or both. For example, a polynucleotide or apolypeptide naturally present in a living organism is not “isolated,”but the same polynucleotide or polypeptide separated from the coexistingmaterials of its natural state is “isolated”, as the term is employedherein.

[0030] “Polynucleotide(s)” generally refers to any polyribonucleotide orpolydeoxribonucleotide, which may be unmodified RNA or DNA or modifiedRNA or DNA. “Polynucleotide(s)” include, without limitation, single- anddouble-stranded DNA, DNA that is a mixture of single- anddouble-stranded regions or single-, double- and triple-stranded regions,single- and double-stranded RNA, and RNA that is mixture of single- anddouble-stranded regions, hybrid molecules comprising DNA and RNA thatmay be single-stranded or, more typically, double-stranded, ortriple-stranded regions, or a mixture of single- and double-strandedregions. In addition, “polynucleotide” as used herein refers totriple-stranded regions comprising RNA or DNA or both RNA and DNA. Thestrands in such regions may be from the same molecule or from differentmolecules. The regions may include all of one or more of the molecules,but more typically involve only a region of some of the molecules. Oneof the molecules of a triple-helical region often is an oligonucleotide.As used herein, the term “polynucleotide(s)” also includes DNAs or RNAsas described above that contain one or more modified bases. Thus, DNAsor RNAs with backbones modified for stability or for other reasons are“polynucleotide(s)” as that term is intended herein. Moreover, DNAs orRNAs comprising unusual bases, such as inosine, or modified bases, suchas tritylated bases, to name just two examples, are polynucleotides asthe term is used herein. It will be appreciated that a great variety ofmodifications have been made to DNA and RNA that serve many usefulpurposes known to those of skill in the art. The term“polynucleotide(s)” as it is employed herein embraces such chemically,enzymatically or metabolically modified forms of polynucleotides, aswell as the chemical forms of DNA and RNA characteristic of viruses andcells, including, for example, simple and complex cells.“Polynucleotide(s)” also embraces short polynucleotides often referredto as oligonucleotide(s).

[0031] “Polypeptide(s)” refers to any peptide or protein comprising twoor more amino acids joined to each other by peptide bonds or modifiedpeptide bonds. “Polypeptide(s)” refers to both short chains, commonlyreferred to as peptides, oligopeptides and oligomers and to longerchains generally referred to as proteins. Polypeptides may contain aminoacids other than the 20 gene encoded amino acids. “Polypeptide(s)”include those modified either by natural processes, such as processingand other post-translational modifications, but also by chemicalmodification techniques. Such modifications are well described in basictexts and in more detailed monographs, as well as in a voluminousresearch literature, and they are well known to those of skill in theart. It will be appreciated that the same type of modification may bepresent in the same or varying degree at several sites in a givenpolypeptide. Also, a given polypeptide may contain many types ofmodifications. Modifications can occur anywhere in a polypeptide,including the peptide backbone, the amino acid side-chains, and theamino or carboxyl termini. Modifications include, for example,acetylation, acylation, ADP-ribosylation, amidation, covalent attachmentof flavin, covalent attachment of a heme moiety, covalent attachment ofa nucleotide or nucleotide derivative, covalent attachment of a lipid orlipid derivative, covalent attachment of phosphotidylinositol,cross-linking, cyclization, disulfide bond formation, demethylation,formation of covalent cross-links, formation of cysteine, formation ofpyroglutamate, formylation, gamma-carboxylation, glycosylation, GPIanchor formation, hydroxylation, iodination, methylation,myristoylation, oxidation, proteolytic processing, phosphorylation,prenylation, racemization, glycosylation, lipid attachment, sulfation,gamma-carboxylation of glutamic acid residues, hydroxylation andADP-ribosylation, selenoylation, sulfation, transfer-RNA mediatedaddition of amino acids to proteins, such as arginylation, andubiquitination. See, for instance, PROTEINS—STRUCTURE AND MOLECULARPROPERTIES, 2nd Ed., T. E. Creighton, W. H. Freeman and Company, NewYork (1993) and Wold, F., Posttranslational Protein Modifications:Perspectives and Prospects, pgs. 1-12 in POSTTRANSLATIONAL COVALENTMODIFICATION OF PROTEINS, B. C. Johnson, Ed., Academic Press, New York(1983); Seifter et al., Meth. Enzymol. 182:626-646 (1990) and Rattan etal., Protein Synthesis: Posttranslational Modifications and Aging, Ann.N.Y. Acad. Sci. 663: 48-62 (1992). Polypeptides may be branched orcyclic, with or without branching. Cyclic, branched and branchedcircular polypeptides may result from post-translational naturalprocesses and may be made by entirely synthetic methods, as well.

[0032] “Variant(s)” as the term is used herein, is a polynucleotide orpolypeptide that differs from a reference polynucleotide or polypeptiderespectively, but retains essential properties. A typical variant of apolynucleotide differs in nucleotide sequence from another, referencepolynucleotide. Changes in the nucleotide sequence of the variant may ormay not alter the amino acid sequence of a polypeptide encoded by thereference polynucleotide. Nucleotide changes may result in amino acidsubstitutions, additions, deletions, fusions and truncations in thepolypeptide encoded by the reference sequence, as discussed below. Atypical variant of a polypeptide differs in amino acid sequence fromanother, reference polypeptide. Generally, differences are limited sothat the sequences of the reference polypeptide and the variant areclosely similar overall and, in many regions, identical. A variant andreference polypeptide may differ in amino acid sequence by one or moresubstitutions, additions, deletions in any combination. A substituted orinserted amino acid residue may or may not be one encoded by the geneticcode. A variant of a polynucleotide or polypeptide may be a naturallyoccurring such as an allelic variant, or it may be a variant that is notknown to occur naturally. Non-naturally occurring variants ofpolynucleotides and polypeptides may be made by mutagenesis techniques,by direct synthesis, and by other recombinant methods known to skilledartisans.

DESCRIPTION OF THE INVENTION

[0033] The invention relates to novel glucosaminidase polypeptides andpolynucleotides as described in greater detail below. In particular, theinvention relates to polypeptides and polynucleotides of a novelglucosaminidase of Staphylococcus aureus, which is related by amino acidsequence homology to sp|P52081|ATL_STAAU AUTOLYSIN PRECURSORpolypeptide. The invention relates especially to glucosaminidase havingthe nucleotide and amino acid sequences set out in Table 1 [SEQ ID NO:1] and Table 1 [SEQ ID NO: 2] respectively, and to the glucosaminidasenucleotide sequences of the DNA in the deposited strain and amino acidsequences encoded thereby. TABLE 1 glucosaminidase Polynucleotide andPolypeptide Sequences (A) Sequences from Staphylococcus aureusglucosaminidase polynucleotide sequence [SEQ ID NO:1]. 5′-1 GGTTTTGTCTATATGAAGAA GAATTTCAAG TTACGCATTT CAACGCTACT  51 ATTGATAGTT ATTTTAGTTGTTTTTGCTGT ATTACTCATC GTGAATGAAA 101 CTAAATTGTT TAAAAATGAT GTGAATTACTCTTTTGATGA GGCTGTTTCA 151 ATGCAACAAG GGAAAGGTAT TGTACAGACA AAAGAAGAGGATGGTAAATT 201 TGTTGAAGCA AATAATAATG AAATTGCTAA AGCAATGACT ATTTCACATA251 AAGACAATGA TATGAAGTAT ATGGATATCA CAGAAAAAGT GCCAATGTCG 301GAATCTGAAG TTAACCAATT GCTAAAAGGT AAGGGGATTT TAGAAAATCG 351 AGGGAAAGTTTTTCTAGAAG CTCAAGAAAA ATATGAGGTT AATGTCATTT 401 ATCTTGTTAG CCATGCATTAGTAGAAACAG GTAACGGCAA ATCAGAATTA 451 GCAAAAGGCA TTAAAGATGG GAAAAAACGCTATTACAACT TTTTTGGTAT 501 AGGAGCATTC GATAGTAGTG CTGTTCGTAG TGGGAAAAGTTATGCTGAAA 551 AGGAACAATG GACATCACCA GATAAGGCGA TTATTGGTGG TGCAAAGTTC601 ATTCGTAATG AATATTTTGA AAACAATCAA CTGAATTTAT ATCAAATGCG 651ATGGAATCCA GAAAATCCTG CGCAACATCA ATATGCGAGT GACATTCGCT 701 GGGCAGATAAAATTGCCAAA TTAATGGATA AATCCTATAA GCAGTTTGGT 751 ATAAAGAAAG ATGATATTAGACAAACATAT TATAAATAA -3′ (B) glucosaminidase polypeptide sequencededuced from the polynucleotide sequence in this table [SEQ ID NO:2].NH₂-1 GFVYMKKNFK LRISTLLLIV ILVVFAVLLI VNETKLFKND VNYSFDEAVS  51MQQGKGIVQT KEEDGKFVEA NNNEIAKAMT ISHKDNDMKY MDITEKVPMS 101 ESEVNQLLKGKGILENRGKV FLEAQEKYEV NVIYLVSHAL VETGNGKSEL 151 AKGIKDGKKR YYNFFGIGAFDSSAVRSGKS YAEKEQWTSP DKAIIGGAKF 201 IRNEYFENNQ LNLYQMRWNP ENPAQHQYASDIRWADKIAK LMDKSYKQFG 251 IKKDDIRQTY YK-COOH (C) Polynucleotide sequenceembodiments [SEQ ID NO: 1]. X-(R₁)_(n)-1 GGTTTTGTCT ATATGAAGAAGAATTTCAAG TTACGCATTT CAACGCTACT  51 ATTGATAGTT ATTTTAGTTG TTTTTGCTGTATTACTCATC GTGAATGAAA 101 CTAAATTGTT TAAAAATGAT GTGAATTACT CTTTTGATGAGGCTGTTTCA 151 ATGCAACAAG GGAAAGGTAT TGTACAGACA AAAGAAGAGG ATGGTAAATT201 TGTTGAAGCA AATAATAATG AAATTGCTAA AGCAATGACT ATTTCACATA 251AAGACAATGA TATGAAGTAT ATGGATATCA CAGAAAAAGT GCCAATGTCG 301 GAATCTGAAGTTAACCAATT GCTAAAAGGT AAGGGGATTT TAGAAAATCG 351 AGGGAAAGTT TTTCTAGAAGCTCAAGAAAA ATATGAGGTT AATGTCATTT 401 ATCTTGTTAG CCATGCATTA GTAGAAACAGGTAACGGCAA ATCAGAATTA 451 GCAAAAGGCA TTAAAGATGG GAAAAAACGC TATTACAACTTTTTTGGTAT 501 AGGAGCATTC GATAGTAGTG CTGTTCGTAG TGGGAAAAGT TATGCTGAAA551 AGGAACAATG GACATCACCA GATAAGGCGA TTATTGGTGG TGCAAAGTTC 601ATTCGTAATG AATATTTTGA AAACAATCAA CTGAATTTAT ATCAAATGCG 651 ATGGAATCCAGAAAATCCTG CGCAACATCA ATATGCGAGT GACATTCGCT 701 GGGCAGATAA AATTGCCAAATTAATGGATA AATCCTATAA GCAGTTTGGT 751 ATAAAGAAAG ATGATATTAG ACAAACATATTATAAATAA -(R₂)_(n)-Y (D) Polypeptide sequence embodiments [SEQ IDNO:2]. X-(R₁)_(n)-1 GFVYMKKNFK LRISTLLLIV TLVVFAVLLI VNETKLFKNDVNYSFDEAVS  51 MQQGKGIVQT KEEDGKFVEA NNNEIAKAMT ISHKDNDMKY MDITEKVPMS101 ESEVNQLLKG KGILENRGKV FLEAQEKYEV NVIYLVSHAL VETGNGKSEL 151AKGIKDGKKR YYNFFGIGAF DSSAVRSGKS YAEKEQWTSP DKAIIGGAKF 201 IRNEYFENNQLNLYQMRWNP ENPAQHQYAS DIRWADKIAK LMDKSYKQFG 251 IKKDDTRQTY YK-(R₂)_(n)-Y(E) Sequences from Staphylococcus aureus glucosaminidase polynucleotideORF sequence [SEQ ID NO:3]. 5′-1 GGTTTTGTCT ATATGAAGAA GAATTTCAAGTTACGCATTT CAACGCTACT  51 ATTGATAGTT ATTTTAGTTG TTTTTGCTGT ATTACTCATCGTGAATGAAA 101 CTAAATTGTT TAAAAATGAT GTGAATTACT CTTTTGATGA GGCTGTTTCA151 ATGCAACAAG GGAAAGGTAT TGTACAGACA AAAGAAGAGG ATGGTAAATT 201TGTTGAAGCA AATAATAATG AAATTGCTAA AGCAATGACT ATTTCACATA 251 AAGACAATGATATGAAGTAT ATGGATATCA CAGAAAAAGT GCCAATGTCG 301 GAATCTGAAG TTAACCAATTGCTAAAAGGT AAGGGGATTT TAGAAAATCG 351 AGGGAAAGTT TTTCTAGAAG CTCAAGAAAAATATGAGGTT AATGTCATTT 401 ATCTTGTTAG CCATGCATTA GTAGAAACAG GTAACGGCAAATCAGAATTA 451 GCAAAAGGCA TTAAAGATGG GAAAAAACGC TATTACAACT TTTTTGGTAT501 AGGAGCATTC GATAGTAGTG CTGTTCGTAG TGGGAAAAGT TATGCTGAAA 551AGGAACAATG GACATCACCA GATAAGGCGA TTATTGGTGG TGCAAAGTTC 601 ATTCGTAATGAATATTTTGA AAACAATCAA CTGAATTTAT ATCAAATGCG 651 ATGGAATCCA GAAAATCCTGCGCAACATCA ATATGCGAGT GACATTCGCT 701 GGGCAGATAA AATTGCCAAA TTAATGGATAAATCCTATAA GCAGTTTGGT 751 ATAAAGAAAG ATGATATTAG ACAAACATAT TATAAA-3′ (F)glucosaminidase polypeptide sequence deduced from the polynucleotide ORFsequence in this table [SEQ ID NO:4]. NH₂-1 GFVYMKKNFK LRISTLLLIVTLVVFAVLLI VNETKLFKND VNYSFDEAVS  51 MQQGKGIVQT KEEDGKFVEA NNNEIAKAMTISHKDNDMKY MDITEKVPMS 101 ESEVNQLLKG KGILENRGKV FLEAQEKYEV NVIYLVSHALVETGNGKSEL 151 AKGIKDGKKR YYNFFGIGAF DSSAVRSGKS YAEKEQWTSP DKAIIGGAKF201 IRNEYFENNQ LNLYQMRWNP ENPAQHQYAS DIRWADKIAK LMDKSYKQFG 251IKKDDIRQTY YK-COOH

Deposited Materials

[0034] A deposit containing a Staphylococcus aureus WCUH 29 strain hasbeen deposited with the National Collections of Industrial and MarineBacteria Ltd. (herein “NCIMB”), 23 St. Machar Drive, Aberdeen AB2 1RY,Scotland on Sep. 11, 1995 and assigned NCIMB Deposit No. 40771, and isreferred to as Staphylococcus aureus WCUH29 on deposit. TheStaphylococcus aureus strain deposit is referred to herein as “thedeposited strain” or as “the DNA of the deposited strain.”

[0035] The deposited strain contains the full length glucosaminidasegene. The sequence of the polynucleotides contained in the depositedstrain, as well as the amino acid sequence of the polypeptide encodedthereby, are controlling in the event of any conflict with anydescription of sequences herein.

[0036] The deposit of the deposited strain has been made under the termsof the Budapest Treaty on the International Recognition of the Depositof Micro-organisms for Purposes of Patent Procedure. The strain will beirrevocably and without restriction or condition released to the publicupon the issuance of a patent. The deposited strain is provided merelyas convenience to those of skill in the art and is not an admission thata deposit is required for enablement, such as that required under 35U.S.C. §112.

[0037] A license may be required to make, use or sell the depositedstrain, and compounds derived therefrom, and no such license is herebygranted.

Polypeptides

[0038] The polypeptides of the invention include the polypeptide ofTable 1 [SEQ ID NO:2] (in particular the mature polypeptide) as well aspolypeptides and fragments, particularly those which have the biologicalactivity of glucosaminidase, and also those which have at least 70%identity to a polypeptide of Table 1 [SEQ ID NOS:2 and 4] or therelevant portion, preferably at least 80% identity to a polypeptide ofTable 1 [SEQ ID NOS:2 and 4], and more preferably at least 90%similarity (more preferably at least 90% identity) to a polypeptide ofTable 1 [SEQ ID NOS:2 and 4] and still more preferably at least 95%similarity (still more preferably at least 95% identity) to apolypeptide of Table 1 [SEQ ID NOS:2 and 4] and also include portions ofsuch polypeptides with such portion of the polypeptide generallycontaining at least 30 amino acids and more preferably at least 50 aminoacids.

[0039] The invention also includes polypeptides of the formula set forthin Table 1 (D) [SEQ ID NO:2] wherein, at the amino terminus, X ishydrogen, and at the carboxyl terminus, Y is hydrogen or a metal, R₁ andR₂ is any amino acid residue, and n is an integer between 1 and 1000.Any stretch of amino acid residues denoted by either R group, where R isgreater than 1, may be either a heteropolymer or a homopolymer,preferably a heteropolymer.

[0040] A fragment is a variant polypeptide having an amino acid sequencethat entirely is the same as part but not all of the amino acid sequenceof the aforementioned polypeptides. As with glucosaminidase polypeptidesfragments may be “free-standing,” or comprised within a largerpolypeptide of which they form a part or region, most preferably as asingle continuous region, a single larger polypeptide.

[0041] Preferred fragments include, for example, truncation polypeptideshaving a portion of an amino acid sequence of Table 1 [SEQ ID NOS:2 and4], or of variants thereof, such as a continuous series of residues thatincludes the amino terminus, or a continuous series of residues thatincludes the carboxyl terminus. Degradation forms of the polypeptides ofthe invention in a host cell, particularly a Staphylococcus aureus, arealso preferred. Further preferred are fragments characterized bystructural or functional attributes such as fragments that comprisealpha-helix and alpha-helix forming regions, beta-sheet andbeta-sheet-forming regions, turn and turn-forming regions, coil andcoil-forming regions, hydrophilic regions, hydrophobic regions, alphaamphipathic regions, beta amphipathic regions, flexible regions,surface-forming regions, substrate binding region, and high antigenicindex regions.

[0042] Also preferred are biologically active fragments which are thosefragments that mediate activities of glucosaminidase, including thosewith a similar activity or an improved activity, or with a decreasedundesirable activity. Also included are those fragments that areantigenic or immunogenic in an animal, especially in a human.Particularly preferred are fragments comprising receptors or domains ofenzymes that confer a function essential for viability of Staphylococcusaureus or the ability to initiate, or maintain cause disease in anindividual, particularly a human.

[0043] Variants that are fragments of the polypeptides of the inventionmay be employed for producing the corresponding full-length polypeptideby peptide synthesis; therefore, these variants may be employed asintermediates for producing the full-length polypeptides of theinvention.

Polynucleotides

[0044] Another aspect of the invention relates to isolatedpolynucleotides, including the full length gene, that encode theglucosaminidase polypeptide having a deduced amino acid sequence ofTable 1 [SEQ ID NOS:2 and 4] and polynucleotides closely related theretoand variants thereof.

[0045] Using the information provided herein, such as a polynucleotidesequence set out in Table 1 [SEQ ID NOS:1 and 3], a polynucleotide ofthe invention encoding glucosaminidase polypeptide may be obtained usingstandard cloning and screening methods, such as those for cloning andsequencing chromosomal DNA fragments from bacteria using Staphylococcusaureus WCUH 29 cells as starting material, followed by obtaining a fulllength clone. For example, to obtain a polynucleotide sequence of theinvention, such as a sequence given in Table 1 [SEQ ID NOS:1 and 3],typically a library of clones of chromosomal DNA of Staphylococcusaureus WCUH 29 in E. coli or some other suitable host is probed with aradiolabeled oligonucleotide, preferably a 17-mer or longer, derivedfrom a partial sequence. Clones carrying DNA identical to that of theprobe can then be distinguished using stringent conditions. Bysequencing the individual clones thus identified with sequencing primersdesigned from the original sequence it is then possible to extend thesequence in both directions to determine the full gene sequence.Conveniently, such sequencing is performed using denatured doublestranded DNA prepared from a plasmid clone. Suitable techniques aredescribed by Maniatis, T., Fritsch, E. F. and Sambrook et al., MOLECULARCLONING, A LABORATORY MANUAL, 2nd Ed.; Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y. (1989). (see in particular Screening ByHybridization 1.90 and Sequencing Denatured Double-Stranded DNATemplates 13.70). Illustrative of the invention, the polynucleotide setout in Table 1 [SEQ ID NO:1] was discovered in a DNA library derivedfrom Staphylococcus aureus WCUH 29.

[0046] The DNA sequence set out in Table 1 [SEQ ID NOS:1] contains anopen reading frame encoding a protein having about the number of aminoacid residues set forth in Table 1 [SEQ ID NO:2] with a deducedmolecular weight that can be calculated using amino acid residuemolecular weight values well known in the art. The polynucleotide of SEQID NO: 1, between nucleotide number 1 through number 786 encodes thepolypeptide of SEQ ID NO:2. The stop codon begins at nucleotide number787 of SEQ ID NO: 1.

[0047] The glucosaminidase polypeptide of the invention is structurallyrelated to other proteins of the autolysin family, as shown by theresults of sequencing the DNA encoding glucosaminidase of the depositedstrain. The protein exhibits greatest homology to sp|P52081|ATL_STAAUAUTOLYSIN PRECURSOR protein among known proteins (see also, autolysinAtlE (GENBANK: locus SEU71377, accession U71377). The glucosaminidasepolypeptide of Table 1 [SEQ ID NO:2] has about 45% identity over itsentire length and about 70% similarity over its entire length with theamino acid sequence of sp|P52081|ATL_STAAU AUTOLYSIN PRECURSORpolypeptide.

[0048] The invention provides a polynucleotide sequence identical overits entire length to the coding sequence in Table 1 [SEQ ID NO:1]. Alsoprovided by the invention is the coding sequence for the maturepolypeptide or a fragment thereof, by itself as well as the codingsequence for the mature polypeptide or a fragment in reading frame withother coding sequence, such as those encoding a leader or secretorysequence, a pre-, or pro- or prepro- protein sequence. Thepolynucleotide may also contain non-coding sequences, including forexample, but not limited to non-coding 5′ and 3′ sequences, such as thetranscribed, non-translated sequences, termination signals, ribosomebinding sites, sequences that stabilize mRNA, introns, polyadenylationsignals, and additional coding sequence which encode additional aminoacids. For example, a marker sequence that facilitates purification ofthe fused polypeptide can be encoded. In certain embodiments of theinvention, the marker sequence is a hexa-histidine peptide, as providedin the pQE vector (Qiagen, Inc.) and described in Gentz et al., Proc.Natl. Acad. Sci., USA 86: 821-824 (1989), or an HA tag (Wilson et al.,Cell 37: 767 (1984). Polynucleotides of the invention also include, butare not limited to, polynucleotides comprising a structural gene and itsnaturally associated sequences that control gene expression.

[0049] A preferred embodiment of the invention is a polynucleotide ofcomprising nucleotide 1 to 786 or 789 set forth in SEQ ID NO:1 of Table1 which encode the glucosaminidase polypeptide.

[0050] The invention also includes polynucleotides of the formula setforth in Table 1 (C)[SEQ ID NO:1] wherein, at the 5′ end of themolecule, X is hydrogen, and at the 3′ end of the molecule, Y ishydrogen or a metal, R₁ and R₂ is any nucleic acid residue, and n is aninteger between 1 and 1000. Any stretch of nucleic acid residues denotedby either R group, where R is greater than 1, may be either aheteropolymer or a homopolymer, preferably a heteropolymer.

[0051] The term “polynucleotide encoding a polypeptide” as used hereinencompasses polynucleotides that include a sequence encoding apolypeptide of the invention, particularly a bacterial polypeptide andmore particularly a polypeptide of the Staphylococcus aureusglucosaminidase having the amino acid sequence set out in Table 1 [SEQID NO:2]. The term also encompasses polynucleotides that include asingle continuous region or discontinuous regions encoding thepolypeptide (for example, interrupted by integrated phage or aninsertion sequence or editing) together with additional regions, thatalso may contain coding and/or non-coding sequences.

[0052] The invention further relates to variants of the polynucleotidesdescribed herein that encode for variants of the polypeptide having thededuced amino acid sequence of Table 1 [SEQ ID NO:2]. Variants that arefragments of the polynucleotides of the invention may be used tosynthesize full-length polynucleotides of the invention.

[0053] Further particularly preferred embodiments are polynucleotidesencoding glucosaminidase variants, that have the amino acid sequence ofglucosaminidase polypeptide of Table 1 [SEQ ID NO:2] in which several, afew, 5 to 10, 1 to 5, 1 to 3, 2, 1 or no amino acid residues aresubstituted, deleted or added, in any combination. Especially preferredamong these are silent substitutions, additions and deletions, that donot alter the properties and activities of glucosaminidase.

[0054] Further preferred embodiments of the invention arepolynucleotides that are at least 70% identical over their entire lengthto a polynucleotide encoding glucosaminidase polypeptide having an aminoacid sequence set out in Table 1 [SEQ ID NOS:2 and 4], andpolynucleotides that are complementary to such polynucleotides.Alternatively, most highly preferred are polynucleotides that comprise aregion that is at least 80% identical over its entire length to apolynucleotide encoding glucosaminidase polypeptide of the depositedstrain and polynucleotides complementary thereto. In this regard,polynucleotides at least 90% identical over their entire length to thesame are particularly preferred, and among these particularly preferredpolynucleotides, those with at least 95% are especially preferred.Furthermore, those with at least 97% are highly preferred among thosewith at least 95%, and among these those with at least 98% and at least99% are particularly highly preferred, with at least 99% being the morepreferred.

[0055] Preferred embodiments are polynucleotides that encodepolypeptides that retain substantially the same biological function oractivity as the mature polypeptide encoded by the DNA of Table 1 [SEQ IDNO:1].

[0056] The invention further relates to polynucleotides that hybridizeto the herein above-described sequences. In this regard, the inventionespecially relates to polynucleotides that hybridize under stringentconditions to the herein above-described polynucleotides. As hereinused, the terms “stringent conditions” and “stringent hybridizationconditions” mean hybridization will occur only if there is at least 95%and preferably at least 97% identity between the sequences. An exampleof stringent hybridization conditions is overnight incubation at 42° C.in a solution comprising: 50% formamide, 5× SSC (150 mM NaCl, 15 mMtrisodium citrate), 50 mM sodium phosphate (pH7.6), 5× Denhardt'ssolution, 10% dextran sulfate, and 20 micrograms/ml denatured, shearedsalmon sperm DNA, followed by washing the hybridization support in 0.1×SSC at about 65° C. Hybridization and wash conditions are well known andexemplified in Sambrook, et al., Molecular Cloning: A Laboratory Manual,Second Edition, Cold Spring Harbor, N.Y., (1989), particularly Chapter11 therein.

[0057] The invention also provides a polynucleotide consistingessentially of a polynucleotide sequence obtainable by screening anappropriate library containing the complete gene for a polynucleotidesequence set forth in SEQ ID NO:1 or SEQ ID NO:3 under stringenthybridization conditions with a probe having the sequence of saidpolynucleotide sequence set forth in SEQ ID NO:1 or a fragment thereof;and isolating said DNA sequence. Fragments useful for obtaining such apolynucleotide include, for example, probes and primers describedelsewhere herein.

[0058] As discussed additionally herein regarding polynucleotide assaysof the invention, for instance, polynucleotides of the invention asdiscussed above, may be used as a hybridization probe for RNA, cDNA andgenomic DNA to isolate full-length cDNAs and genomic clones encodingglucosaminidase and to isolate cDNA and genomic clones of other genesthat have a high sequence similarity to the glucosaminidase gene. Suchprobes generally will comprise at least 15 bases. Preferably, suchprobes will have at least 30 bases and may have at least 50 bases.Particularly preferred probes will have at least 30 bases and will have50 bases or less.

[0059] For example, the coding region of the glucosaminidase gene may beisolated by screening using the DNA sequence provided in SEQ ID NO:1 tosynthesize an oligonucleotide probe. A labeled oligonucleotide having asequence complementary to that of a gene of the invention is then usedto screen a library of cDNA, genomic DNA or mRNA to determine whichmembers of the library the probe hybridizes to.

[0060] The polynucleotides and polypeptides of the invention may beemployed, for example, as research reagents and materials for discoveryof treatments of and diagnostics for disease, particularly humandisease, as further discussed herein relating to polynucleotide assays.

[0061] Polynucleotides of the invention that are oligonucleotidesderived from the sequences of SEQ ID NOS:1 and/or 2 may be used in theprocesses herein as described, but preferably for PCR, to determinewhether or not the polynucleotides identified herein in whole or in partare transcribed in bacteria in infected tissue. It is recognized thatsuch sequences will also have utility in diagnosis of the stage ofinfection and type of infection the pathogen has attained.

[0062] The invention also provides polynucleotides that may encode apolypeptide that is the mature protein plus additional amino orcarboxyl-terminal amino acids, or amino acids interior to the maturepolypeptide (when the mature form has more than one polypeptide chain,for instance). Such sequences may play a role in processing of a proteinfrom precursor to a mature form, may allow protein transport, maylengthen or shorten protein half-life or may facilitate manipulation ofa protein for assay or production, among other things. As generally isthe case in vivo, the additional amino acids may be processed away fromthe mature protein by cellular enzymes.

[0063] A precursor protein, having the mature form of the polypeptidefused to one or more prosequences may be an inactive form of thepolypeptide. When prosequences are removed such inactive precursorsgenerally are activated. Some or all of the prosequences may be removedbefore activation. Generally, such precursors are called proproteins.

[0064] In sum, a polynucleotide of the invention may encode a matureprotein, a mature protein plus a leader sequence (which may be referredto as a preprotein), a precursor of a mature protein having one or moreprosequences that are not the leader sequences of a preprotein, or apreproprotein, which is a precursor to a proprotein, having a leadersequence and one or more prosequences, which generally are removedduring processing steps that produce active and mature forms of thepolypeptide.

Vectors, Host Cells, Expression

[0065] The invention also relates to vectors that comprise apolynucleotide or polynucleotides of the invention, host cells that aregenetically engineered with vectors of the invention and the productionof polypeptides of the invention by recombinant techniques. Cell-freetranslation systems can also be employed to produce such proteins usingRNAs derived from the DNA constructs of the invention.

[0066] For recombinant production, host cells can be geneticallyengineered to incorporate expression systems or portions thereof orpolynucleotides of the invention. Introduction of a polynucleotide intothe host cell can be effected by methods described in many standardlaboratory manuals, such as Davis et al., BASIC METHODS IN MOLECULARBIOLOGY, (1986) and Sambrook et al., MOLECULAR CLONING: A LABORATORYMANUAL, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y. (1989), such as, calcium phosphate transfection,DEAE-dextran mediated transfection, transvection, microinjection,cationic lipid-mediated transfection, electroporation, transduction,scrape loading, ballistic introduction and infection.

[0067] Representative examples of appropriate hosts include bacterialcells, such as streptococci, staphylococci, enterococci E. coli,streptomyces and Bacillus subtilis cells; fungal cells, such as yeastcells and Aspergillus cells; insect cells such as Drosophila S2 andSpodoptera Sf9 cells; animal cells such as CHO, COS, HeLa, C127, 3T3,BHK, 293 and Bowes melanoma cells; and plant cells.

[0068] A great variety of expression systems can be used to produce thepolypeptides of the invention. Such vectors include, among others,chromosomal, episomal and virus-derived vectors, e.g., vectors derivedfrom bacterial plasmids, from bacteriophage, from transposons, fromyeast episomes, from insertion elements, from yeast chromosomalelements, from viruses such as baculoviruses, papova viruses, such asSV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabiesviruses and retroviruses, and vectors derived from combinations thereof,such as those derived from plasmid and bacteriophage genetic elements,such as cosmids and phagemids. The expression system constructs maycontain control regions that regulate as well as engender expression.Generally, any system or vector suitable to maintain, propagate orexpress polynucleotides and/or to express a polypeptide in a host may beused for expression in this regard. The appropriate DNA sequence may beinserted into the expression system by any of a variety of well-knownand routine techniques, such as, for example, those set forth inSambrook et al., MOLECULAR CLONING, A LABORATORY MANUAL, (supra).

[0069] For secretion of the translated protein into the lumen of theendoplasmic reticulum, into the periplasmic space or into theextracellular environment, appropriate secretion signals may beincorporated into the expressed polypeptide. These signals may beendogenous to the polypeptide or they may be heterologous signals.

[0070] Polypeptides of the invention can be recovered and purified fromrecombinant cell cultures by well-known methods including ammoniumsulfate or ethanol precipitation, acid extraction, anion or cationexchange chromatography, phosphocellulose chromatography, hydrophobicinteraction chromatography, affinity chromatography, hydroxylapatitechromatography, and lectin chromatography. Most preferably, highperformance liquid chromatography is employed for purification. Wellknown techniques for refolding protein may be employed to regenerateactive conformation when the polypeptide is denatured during isolationand or purification.

Diagnostic Assays

[0071] This invention is also related to the use of the glucosaminidasepolynucleotides of the invention for use as diagnostic reagents.Detection of glucosaminidase in a eukaryote, particularly a mammal, andespecially a human, will provide a diagnostic method for diagnosis of adisease. Eukaryotes (herein also “individual(s)”), particularly mammals,and especially humans, infected with an organism comprising theglucosaminidase gene may be detected at the nucleic acid level by avariety of techniques.

[0072] Nucleic acids for diagnosis may be obtained from an infectedindividual's cells and tissues, such as bone, blood, muscle, cartilage,and skin. Genomic DNA may be used directly for detection or may beamplified enzymatically by using PCR or other amplification techniqueprior to analysis. RNA or cDNA may also be used in the same ways. Usingamplification, characterization of the species and strain of prokaryotepresent in an individual, may be made by an analysis of the genotype ofthe prokaryote gene. Deletions and insertions can be detected by achange in size of the amplified product in comparison to the genotype ofa reference sequence. Point mutations can be identified by hybridizingamplified DNA to labeled glucosaminidase polynucleotide sequences.Perfectly matched sequences can be distinguished from mismatchedduplexes by RNase digestion or by differences in melting temperatures.DNA sequence differences may also be detected by alterations in theelectrophoretic mobility of the DNA fragments in gels, with or withoutdenaturing agents, or by direct DNA sequencing. See, e.g., Myers et al.,Science, 230: 1242 (1985). Sequence changes at specific locations alsomay be revealed by nuclease protection assays, such as RNase and S1protection or a chemical cleavage method. See, e.g., Cotton et al.,Proc. Natl. Acad. Sci., USA, 85: 4397-4401 (1985).

[0073] Cells carrying mutations or polymorphisms in the gene of theinvention may also be detected at the DNA level by a variety oftechniques, to allow for serotyping, for example. For example, RT-PCRcan be used to detect mutations. It is particularly preferred to usedRT-PCR in conjunction with automated detection systems, such as, forexample, GeneScan. RNA or cDNA may also be used for the same purpose,PCR or RT-PCR. As an example, PCR primers complementary to a nucleicacid encoding glucosaminidase can be used to identify and analyzemutations. Examples of representative primers are shown below in Table2. TABLE 2 Primers for amplification of glucosaminidase polynucleotidesSEQ ID NO PRIMER SEQUENCE 5 5′-G TTAACCAATT GCTAAAAGG-3′ 65′-AAACTGCTTATAGGATTTATC-3′

[0074] The invention further provides these primers with 1, 2, 3 or 4nucleotides removed from the 5′ and/or the 3′ end. These primers may beused for, among other things, amplifying glucosaminidase DNA isolatedfrom a sample derived from an individual. The primers may be used toamplify the gene isolated from an infected individual such that the genemay then be subject to various techniques for elucidation of the DNAsequence. In this way, mutations in the DNA sequence may be detected andused to diagnose infection and to serotype and/or classify theinfectious agent.

[0075] The invention further provides a process for diagnosing, disease,preferably bacterial infections, more preferably infections byStaphylococcus aureus, and most preferably disease, such as, infectionsof the upper respiratory tract (e.g., otitis media, bacterialtracheitis, acute epiglottitis, thyroiditis), lower respiratory (e.g.,empyema, lung abscess), cardiac (e.g., infective endocarditis),gastrointestinal (e.g., secretory diarrhoea, splenic abscess,retroperitoneal abscess), CNS (e.g., cerebral abscess), eye (e.g.,blepharitis, conjunctivitis, keratitis, endophthalmitis, preseptal andorbital cellulitis, darcryocystitis), kidney and urinary tract (e.g.,epididymitis, intrarenal and perinephric abscess, toxic shock syndrome),skin (e.g., impetigo, folliculitis, cutaneous abscesses, cellulitis,wound infection, bacterial myositis) bone and joint (e.g., septicarthritis, osteomyelitis), comprising determining from a sample derivedfrom an individual a increased level of expression of polynucleotidehaving the sequence of Table 1 [SEQ ID NO:1]. Increased or decreasedexpression of glucosaminidase polynucleotide can be measured using anyon of the methods well known in the art for the quantation ofpolynucleotides, such as, for example, amplification, PCR, RT-PCR, RNaseprotection, Northern blotting and other hybridization methods.

[0076] In addition, a diagnostic assay in accordance with the inventionfor detecting over-expression of glucosaminidase protein compared tonormal control tissue samples may be used to detect the presence of aninfection, for example. Assay techniques that can be used to determinelevels of a glucosaminidase protein, in a sample derived from a host arewell-known to those of skill in the art. Such assay methods includeradioimmunoassays, competitive-binding assays, Western Blot analysis andELISA assays.

Antibodies

[0077] The polypeptides of the invention or variants thereof, or cellsexpressing them can be used as an immunogen to produce antibodiesimmunospecific for such polypeptides. “Antibodies” as used hereinincludes monoclonal and polyclonal antibodies, chimeric, single chain,simianized antibodies and humanized antibodies, as well as Fabfragments, including the products of an Fab immunolglobulin expressionlibrary.

[0078] Antibodies generated against the polypeptides of the inventioncan be obtained by administering the polypeptides or epitope-bearingfragments, analogues or cells to an animal, preferably a nonhuman, usingroutine protocols. For preparation of monoclonal antibodies, anytechnique known in the art that provides antibodies produced bycontinuous cell line cultures can be used. Examples include varioustechniques, such as those in Kohler, G. and Milstein, C., Nature 256:495-497 (1975); Kozbor et al., Immunology Today 4: 72 (1983); Cole etal., pg. 77-96 in MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R.Liss, Inc. (1985).

[0079] Techniques for the production of single chain antibodies (U.S.Pat. No. 4,946,778) can be adapted to produce single chain antibodies topolypeptides of this invention. Also, transgenic mice, or otherorganisms such as other mammals, may be used to express humanizedantibodies.

[0080] Alternatively phage display technology may be utilized to selectantibody genes with binding activities towards the polypeptide eitherfrom repertoires of PCR amplified v-genes of lymphocytes from humansscreened for possessing anti-glucosaminidase or from naive libraries(McCafferty, J. et al., (1990), Nature 348, 552-554; Marks, J. et al.,(1992) Biotechnology 10, 779-783). The affinity of these antibodies canalso be improved by chain shuffling (Clackson, T. et al., (1991) Nature352, 624-628).

[0081] If two antigen binding domains are present each domain may bedirected against a different epitope—termed ‘bispecific’ antibodies.

[0082] The above-described antibodies may be employed to isolate or toidentify clones expressing the polypeptides to purify the polypeptidesby affinity chromatography.

[0083] Thus, among others, antibodies against glucosaminidase-polypeptide may be employed to treat infections, particularly bacterialinfections and especially disease, such as, infections of the upperrespiratory tract (e.g., otitis media, bacterial tracheitis, acuteepiglottitis, thyroiditis), lower respiratory (e.g., empyema, lungabscess), cardiac (e.g., infective endocarditis), gastrointestinal(e.g., secretory diarrhoea, splenic abscess, retroperitoneal abscess),CNS (e.g., cerebral abscess), eye (e.g., blepharitis, conjunctivitis,keratitis, endophthalmitis, preseptal and orbital cellulitis,darcryocystitis), kidney and urinary tract (e.g., epididymitis,intrarenal and perinephric abscess, toxic shock syndrome), skin (e.g.,impetigo, folliculitis, cutaneous abscesses, cellulitis, woundinfection, bacterial myositis) bone and joint (e.g., septic arthritis,osteomyelitis).

[0084] Polypeptide variants include antigenically, epitopically orimmunologically equivalent variants that form a particular aspect ofthis invention. The term “antigenically equivalent derivative” as usedherein encompasses a polypeptide or its equivalent which will bespecifically recognized by certain antibodies which, when raised to theprotein or polypeptide according to the invention, interfere with theimmediate physical interaction between pathogen and mammalian host. Theterm “immunologically equivalent derivative” as used herein encompassesa peptide or its equivalent which when used in a suitable formulation toraise antibodies in a vertebrate, the antibodies act to interfere withthe immediate physical interaction between pathogen and mammalian host.

[0085] The polypeptide, such as an antigenically or immunologicallyequivalent derivative or a fusion protein thereof is used as an antigento immunize a mouse or other animal such as a rat or chicken. The fusionprotein may provide stability to the polypeptide. The antigen may beassociated, for example by conjugation, with an immunogenic carrierprotein for example bovine serum albumin (BSA) or keyhole limpethaemocyanin (KLH). Alternatively a multiple antigenic peptide comprisingmultiple copies of the protein or polypeptide, or an antigenically orimmunologically equivalent polypeptide thereof may be sufficientlyantigenic to improve immunogenicity so as to obviate the use of acarrier.

[0086] Preferably, the antibody or variant thereof is modified to makeit less immunogenic in the individual. For example, if the individual ishuman the antibody may most preferably be “humanized”; where thecomplimentarity determining region(s) of the hybridoma-derived antibodyhas been transplanted into a human monoclonal antibody, for example asdescribed in Jones, P. et al. (1986), Nature 321, 522-525 or Tempest etal., (1991) Biotechnology 9, 266-273.

[0087] The use of a polynucleotide of the invention in geneticimmunization will preferably employ a suitable delivery method such asdirect injection of plasmid DNA into muscles (Wolff et al., Hum MolGenet 1992, 1:363, Manthorpe et al., Hum. Gene Ther. 1963:4, 419),delivery of DNA complexed with specific protein carriers (Wu et al., JBiol Chem. 1989: 264,16985), coprecipitation of DNA with calciumphosphate (Benvenisty & Reshef, PNAS USA, 1986:83,9551), encapsulationof DNA in various forms of liposomes (Kaneda et al., Science1989:243,375), particle bombardment (Tang et al., Nature 1992, 356:152,Eisenbraun et al., DNA Cell Biol 1993, 12:791) and in vivo infectionusing cloned retroviral vectors (Seeger et al., PNAS USA 1984:81,5849).

Antagonists and Agonists—Assays and Molecules

[0088] Polypeptides of the invention may also be used to assess thebinding of small molecule substrates and ligands in, for example, cells,cell-free preparations, chemical libraries, and natural productmixtures. These substrates and ligands may be natural substrates andligands or may be structural or functional mimetics. See, e.g., Coliganet al., Current Protocols in Immunology 1(2): Chapter 5 (1991).

[0089] The invention also provides a method of screening compounds toidentify those which enhance (agonist) or block (antagonist) the actionof glucosaminidase polypeptides or polynucleotides, particularly thosecompounds that are bacteriostatic and/or bacteriocidal. The method ofscreening may involve high-throughput techniques. For example, to screenfor agonists or antagoists, a synthetic reaction mix, a cellularcompartment, such as a membrane, cell envelope or cell wall, or apreparation of any thereof, comprising glucosaminidase polypeptide and alabeled substrate or ligand of such polypeptide is incubated in theabsence or the presence of a candidate molecule that may be aglucosaminidase agonist or antagonist. The ability of the candidatemolecule to agonize or antagonize the glucosaminidase polypeptide isreflected in decreased binding of the labeled ligand or decreasedproduction of product from such substrate. Molecules that bindgratuitously, i.e., without inducing the effects of glucosaminidasepolypeptide are most likely to be good antagonists. Molecules that bindwell and increase the rate of product production from substrate areagonists. Detection of the rate or level of production of product fromsubstrate may be enhanced by using a reporter system. Reporter systemsthat may be useful in this regard include but are not limited tocolorimetric labeled substrate converted into product, a reporter genethat is responsive to changes in glucosaminidase polynucleotide orpolypeptide activity, and binding assays known in the art.

[0090] Another example of an assay for glucosaminidase antagonists is acompetitive assay that combines glucosaminidase and a potentialantagonist with glucosaminidase-binding molecules, recombinantglucosaminidase binding molecules, natural substrates or ligands, orsubstrate or ligand mimetics, under appropriate conditions for acompetitive inhibition assay. The glucosaminidase molecule can belabeled, such as by radioactivity or a colorimetric compound, such thatthe number of glucosaminidase molecules bound to a binding molecule orconverted to product can be determined accurately to assess theeffectiveness of the potential antagonist.

[0091] Potential antagonists include small organic molecules, peptides,polypeptides and antibodies that bind to a polynucleotide or polypeptideof the invention and thereby inhibit or extinguish its activity.Potential antagonists also may be small organic molecules, a peptide, apolypeptide such as a closely related protein or antibody that binds thesame sites on a binding molecule, such as a binding molecule, withoutinducing glucosaminidase-induced activities, thereby preventing theaction of glucosaminidase by excluding glucosaminidase from binding.

[0092] Potential antagonists include a small molecule that binds to andoccupies the binding site of the polypeptide thereby preventing bindingto cellular binding molecules, such that normal biological activity isprevented. Examples of small molecules include but are not limited tosmall organic molecules, peptides or peptide-like molecules. Otherpotential antagonists include antisense molecules (see Okano, J.Neurochem. 56: 560 (1991); OLIGODEOXYNUCLEOTIDES AS ANTISENSE INHIBITORSOF GENE EXPRESSION, CRC Press, Boca Raton, Fla. (1988), for adescription of these molecules). Preferred potential antagonists includecompounds related to and variants of glucosaminidase.

[0093] Each of the DNA sequences provided herein may be used in thediscovery and development of antibacterial compounds. The encodedprotein, upon expression, can be used as a target for the screening ofantibacterial drugs. Additionally, the DNA sequences encoding the aminoterminal regions of the encoded protein or Shine-Delgarno or othertranslation facilitating sequences of the respective mRNA can be used toconstruct antisense sequences to control the expression of the codingsequence of interest.

[0094] The invention also provides the use of the polypeptide,polynucleotide or inhibitor of the invention to interfere with theinitial physical interaction between a pathogen and mammalian hostresponsible for sequelae of infection. In particular the molecules ofthe invention may be used: in the prevention of adhesion of bacteria, inparticular gram positive bacteria, to mammalian extracellular matrixproteins on in-dwelling devices or to extracellular matrix proteins inwounds; to block glucosaminidase protein-mediated mammalian cellinvasion by, for example, initiating phosphorylation of mammaliantyrosine kinases (Rosenshine et al., Infect. Immun. 60:2211 (1992); toblock bacterial adhesion between mammalian extracellular matrix proteinsand bacterial glucosaminidase proteins that mediate tissue damage and;to block the normal progression of pathogenesis in infections initiatedother than by the implantation of in-dwelling devices or by othersurgical techniques.

[0095] The antagonists and agonists of the invention may be employed,for instance, to inhibit and treat disease, such as, infections of theupper respiratory tract (e.g., otitis media, bacterial tracheitis, acuteepiglottitis, thyroiditis), lower respiratory (e.g., empyema, lungabscess), cardiac (e.g., infective endocarditis), gastrointestinal(e.g., secretory diarrhoea, splenic abscess, retroperitoneal abscess),CNS (e.g., cerebral abscess), eye (e.g., blepharitis, conjunctivitis,keratitis, endophthalmitis, preseptal and orbital cellulitis,darcryocystitis), kidney and urinary tract (e.g., epididymitis,intrarenal and perinephric abscess, toxic shock syndrome), skin (e.g.,impetigo, folliculitis, cutaneous abscesses, cellulitis, woundinfection, bacterial myositis) bone and joint (e.g., septic arthritis,osteomyelitis).

Vaccines

[0096] Another aspect of the invention relates to a method for inducingan immunological response in an individual, particularly a mammal whichcomprises inoculating the individual with glucosaminidase, or a fragmentor variant thereof, adequate to produce antibody and/or T cell immuneresponse to protect said individual from infection, particularlybacterial infection and most particularly Staphylococcus aureusinfection. Also provided are methods whereby such immunological responseslows bacterial replication. Yet another aspect of the invention relatesto a method of inducing immunological response in an individual whichcomprises delivering to such individual a nucleic acid vector to directexpression of glucosaminidase, or a fragment or a variant thereof, forexpressing glucosaminidase, or a fragment or a variant thereof in vivoin order to induce an immunological response, such as, to produceantibody and/or T cell immune response, including, for example,cytokine-producing T cells or cytotoxic T cells, to protect saidindividual from disease, whether that disease is already establishedwithin the individual or not. One way of administering the gene is byaccelerating it into the desired cells as a coating on particles orotherwise.

[0097] Such nucleic acid vector may comprise DNA, RNA, a modifiednucleic acid, or a DNA/RNA hybrid.

[0098] A further aspect of the invention relates to an immunologicalcomposition which, when introduced into an individual capable or havinginduced within it an immunological response, induces an immunologicalresponse in such individual to a glucosaminidase or protein codedtherefrom, wherein the composition comprises a recombinantglucosaminidase or protein coded therefrom comprising DNA which codesfor and expresses an antigen of said glucosaminidase or protein codedtherefrom. The immunological response may be used therapeutically orprophylactically and may take the form of antibody immunity or cellularimmunity such as that arising from CTL or CD4+ T cells.

[0099] A glucosaminidase polypeptide or a fragment thereof may be fusedwith co-protein which may not by itself produce antibodies, but iscapable of stabilizing the first protein and producing a fused proteinwhich will have immunogenic and protective properties. Thus fusedrecombinant protein, preferably further comprises an antigenicco-protein, such as lipoprotein D from Hemophilus influenzae,Glutathione-S-transferase (GST) or beta-galactosidase, relatively largeco-proteins which solubilize the protein and facilitate production andpurification thereof. Moreover, the co-protein may act as an adjuvant inthe sense of providing a generalized stimulation of the immune system.The co-protein may be attached to either the amino or carboxy terminusof the first protein.

[0100] Provided by this invention are compositions, particularly vaccinecompositions, and methods comprising the polypeptides or polynucleotidesof the invention and immunostimulatory DNA sequences, such as thosedescribed in Sato, Y. et al. Science 273: 352 (1996).

[0101] Also, provided by this invention are methods using the describedpolynucleotide or particular fragments thereof which have been shown toencode non-variable regions of bacterial cell surface proteins in DNAconstructs used in such genetic immunization experiments in animalmodels of infection with Staphylococcus aureus will be particularlyuseful for identifying protein epitopes able to provoke a prophylacticor therapeutic immune response. It is believed that this approach willallow for the subsequent preparation of monoclonal antibodies ofparticular value from the requisite organ of the animal successfullyresisting or clearing infection for the development of prophylacticagents or therapeutic treatments of bacterial infection, particularlyStaphylococcus aureus infection, in mammals, particularly humans.

[0102] The polypeptide may be used as an antigen for vaccination of ahost to produce specific antibodies which protect against invasion ofbacteria, for example by blocking adherence of bacteria to damagedtissue. Examples of tissue damage include wounds in skin or connectivetissue caused, e.g., by mechanical, chemical or thermal damage or byimplantation of indwelling devices, or wounds in the mucous membranes,such as the mouth, mammary glands, urethra or vagina.

[0103] The invention also includes a vaccine formulation which comprisesan immunogenic recombinant protein of the invention together with asuitable carrier. Since the protein may be broken down in the stomach,it is preferably administered parenterally, including, for example,administration that is subcutaneous, intramuscular, intravenous, orintradermal. Formulations suitable for parenteral administration includeaqueous and non-aqueous sterile injection solutions which may containanti-oxidants, buffers, bacteriostats and solutes which render theformulation insotonic with the bodily fluid, preferably the blood, ofthe individual; and aqueous and non-aqueous sterile suspensions whichmay include suspending agents or thickening agents. The formulations maybe presented in unit-dose or multi-dose containers, for example, sealedampules and vials and may be stored in a freeze-dried conditionrequiring only the addition of the sterile liquid carrier immediatelyprior to use. The vaccine formulation may also include adjuvant systemsfor enhancing the immunogenicity of the formulation, such as oil-inwater systems and other systems known in the art. The dosage will dependon the specific activity of the vaccine and can be readily determined byroutine experimentation.

[0104] While the invention has been described with reference to certainglucosaminidase protein, it is to be understood that this coversfragments of the naturally occurring protein and similar proteins withadditions, deletions or substitutions which do not substantially affectthe immunogenic properties of the recombinant protein.

Compositions, Kits and Administration

[0105] The invention also relates to compositions comprising thepolynucleotide or the polypeptides discussed above or their agonists orantagonists. The polypeptides of the invention may be employed incombination with a non-sterile or sterile carrier or carriers for usewith cells, tissues or organisms, such as a pharmaceutical carriersuitable for administration to a subject. Such compositions comprise,for instance, a media additive or a therapeutically effective amount ofa polypeptide of the invention and a pharmaceutically acceptable carrieror excipient. Such carriers may include, but are not limited to, saline,buffered saline, dextrose, water, glycerol, ethanol and combinationsthereof. The formulation should suit the mode of administration. Theinvention further relates to diagnostic and pharmaceutical packs andkits comprising one or more containers filled with one or more of theingredients of the aforementioned compositions of the invention.

[0106] Polypeptides and other compounds of the invention may be employedalone or in conjunction with other compounds, such as therapeuticcompounds.

[0107] The pharmaceutical compositions may be administered in anyeffective, convenient manner including, for instance, administration bytopical, oral, anal, vaginal, intravenous, intraperitoneal,intramuscular, subcutaneous, intranasal or intradermal routes amongothers.

[0108] In therapy or as a prophylactic, the active agent may beadministered to an individual as an injectable composition, for exampleas a sterile aqueous dispersion, preferably isotonic.

[0109] Alternatively the composition may be formulated for topicalapplication for example in the form of ointments, creams, lotions, eyeointments, eye drops, ear drops, mouthwash, impregnated dressings andsutures and aerosols, and may contain appropriate conventionaladditives, including, for example, preservatives, solvents to assistdrug penetration, and emollients in ointments and creams. Such topicalformulations may also contain compatible conventional carriers, forexample cream or ointment bases, and ethanol or oleyl alcohol forlotions. Such carriers may constitute from about 1% to about 98% byweight of the formulation; more usually they will constitute up to about80% by weight of the formulation.

[0110] For administration to mammals, and particularly humans, it isexpected that the daily dosage level of the active agent will be from0.01 mg/kg to 10 mg/kg, typically around 1 mg/kg. The physician in anyevent will determine the actual dosage which will be most suitable foran individual and will vary with the age, weight and response of theparticular individual. The above dosages are exemplary of the averagecase. There can, of course, be individual instances where higher orlower dosage ranges are merited, and such are within the scope of thisinvention.

[0111] In-dwelling devices include surgical implants, prosthetic devicesand catheters, i.e., devices that are introduced to the body of anindividual and remain in position for an extended time. Such devicesinclude, for example, artificial joints, heart valves, pacemakers,vascular grafts, vascular catheters, cerebrospinal fluid shunts, urinarycatheters, continuous ambulatory peritoneal dialysis (CAPD) catheters.

[0112] The composition of the invention may be administered by injectionto achieve a systemic effect against relevant bacteria shortly beforeinsertion of an in-dwelling device. Treatment may be continued aftersurgery during the in-body time of the device. In addition, thecomposition could also be used to broaden perioperative cover for anysurgical technique to prevent bacterial wound infections, especiallyStaphylococcus aureus wound infections.

[0113] Many orthopaedic surgeons consider that humans with prostheticjoints should be considered for antibiotic prophylaxis before dentaltreatment that could produce a bacteremia. Late deep infection is aserious complication sometimes leading to loss of the prosthetic jointand is accompanied by significant morbidity and mortality. It maytherefore be possible to extend the use of the active agent as areplacement for prophylactic antibiotics in this situation.

[0114] In addition to the therapy described above, the compositions ofthis invention may be used generally as a wound treatment agent toprevent adhesion of bacteria to matrix proteins exposed in wound tissueand for prophylactic use in dental treatment as an alternative to, or inconjunction with, antibiotic prophylaxis.

[0115] Alternatively, the composition of the invention may be used tobathe an indwelling device immediately before insertion. The activeagent will preferably be present at a concentration of 1 μg/ml to 10mg/ml for bathing of wounds or indwelling devices.

[0116] A vaccine composition is conveniently in injectable form.Conventional adjuvants may be employed to enhance the immune response. Asuitable unit dose for vaccination is 0.5-5 microgram/kg of antigen, andsuch dose is preferably administered 1-3 times and with an interval of1-3 weeks. With the indicated dose range, no adverse toxicologicaleffects will be observed with the compounds of the invention which wouldpreclude their administration to suitable individuals.

[0117] Each reference disclosed herein is incorporated by referenceherein in its entirety. Any patent application to which this applicationclaims priority is also incorporated by reference herein in itsentirety.

EXAMPLES

[0118] The examples below are carried out using standard techniques,which are well known and routine to those of skill in the art, exceptwhere otherwise described in detail. The examples are illustrative, butdo not limit the invention.

Example 1 Strain Selection, Library Production and Sequencing

[0119] The polynucleotide having the DNA sequence given in SEQ ID NO:1was obtained from a library of clones of chromosomal DNA ofStaphylococcus aureus in E. coli. The sequencing data from two or moreclones containing overlapping Staphylococcus aureus DNAs was used toconstruct the contiguous DNA sequence in SEQ ID NO:1. Libraries may beprepared by routine methods, for example: Methods 1 and 2 below.

[0120] Total cellular DNA is isolated from Staphylococcus aureus WCUH 29according to standard procedures and size-fractionated by either of twomethods.

Method 1

[0121] Total cellular DNA is mechanically sheared by passage through aneedle in order to size-fractionate according to standard procedures.DNA fragments of up to 11 kbp in size are rendered blunt by treatmentwith exonuclease and DNA polymerase, and EcoRI linkers added. Fragmentsare ligated into the vector Lambda ZapII that has been cut with EcoRI,the library packaged by standard procedures and E. coli infected withthe packaged library. The library is amplified by standard procedures.

Method 2

[0122] Total cellular DNA is partially hydrolyzed with a one or acombination of restriction enzymes appropriate to generate a series offragments for cloning into library vectors (e.g., RsaI, PalI, AluI,Bsh1235I), and such fragments are size-fractionated according tostandard procedures. EcoRI linkers are ligated to the DNA and thefragments then ligated into the vector Lambda ZapII that have been cutwith EcoRI, the library packaged by standard procedures, and E. coliinfected with the packaged library. The library is amplified by standardprocedures.

1 6 789 base pairs nucleic acid double linear 1 GGTTTTGTCT ATATGAAGAAGAATTTCAAG TTACGCATTT CAACGCTACT ATTGATAGTT 60 ATTTTAGTTG TTTTTGCTGTATTACTCATC GTGAATGAAA CTAAATTGTT TAAAAATGAT 120 GTGAATTACT CTTTTGATGAGGCTGTTTCA ATGCAACAAG GGAAAGGTAT TGTACAGACA 180 AAAGAAGAGG ATGGTAAATTTGTTGAAGCA AATAATAATG AAATTGCTAA AGCAATGACT 240 ATTTCACATA AAGACAATGATATGAAGTAT ATGGATATCA CAGAAAAAGT GCCAATGTCG 300 GAATCTGAAG TTAACCAATTGCTAAAAGGT AAGGGGATTT TAGAAAATCG AGGGAAAGTA 360 TTTCTAGAAG CTCAAGAAAAATATGAGGTT AATGTCATTT ATCTTGTTAG CCATGCATTA 420 GTAGAAACAG GTAACGGCAAATCAGAATTA GCAAAAGGCA TTAAAGATGG GAAAAAACGC 480 TATTACAACT TTTTTGGTATAGGAGCATTC GATAGTAGTG CTGTTCGTAG TGGGAAAAGT 540 TATGCTGAAA AGGAACAATGGACATCACCA GATAAGGCGA TTATTGGTGG TGCAAAGTTC 600 ATTCGTAATG AATATTTTGAAAACAATCAA CTGAATTTAT ATCAAATGCG ATGGAATCCA 660 GAAAATCCTG CGCAACATCAATATGCGAGT GACATTCGCT GGGCAGATAA AATTGCCAAA 720 TTAATGGATA AATCCTATAAGCAGTTTGGT ATAAAGAAAG ATGATATTAG ACAAACATAT 780 TATAAATAA 789 262 aminoacids amino acid single linear 2 Gly Phe Val Tyr Met Lys Lys Asn Phe LysLeu Arg Ile Ser Thr Leu 1 5 10 15 Leu Leu Ile Val Ile Leu Val Val PheAla Val Leu Leu Ile Val Asn 20 25 30 Glu Thr Lys Leu Phe Lys Asn Asp ValAsn Tyr Ser Phe Asp Glu Ala 35 40 45 Val Ser Met Gln Gln Gly Lys Gly IleVal Gln Thr Lys Glu Glu Asp 50 55 60 Gly Lys Phe Val Glu Ala Asn Asn AsnGlu Ile Ala Lys Ala Met Thr 65 70 75 80 Ile Ser His Lys Asp Asn Asp MetLys Tyr Met Asp Ile Thr Glu Lys 85 90 95 Val Pro Met Ser Glu Ser Glu ValAsn Gln Leu Leu Lys Gly Lys Gly 100 105 110 Ile Leu Glu Asn Arg Gly LysVal Phe Leu Glu Ala Gln Glu Lys Tyr 115 120 125 Glu Val Asn Val Ile TyrLeu Val Ser His Ala Leu Val Glu Thr Gly 130 135 140 Asn Gly Lys Ser GluLeu Ala Lys Gly Ile Lys Asp Gly Lys Lys Arg 145 150 155 160 Tyr Tyr AsnPhe Phe Gly Ile Gly Ala Phe Asp Ser Ser Ala Val Arg 165 170 175 Ser GlyLys Ser Tyr Ala Glu Lys Glu Gln Trp Thr Ser Pro Asp Lys 180 185 190 AlaIle Ile Gly Gly Ala Lys Phe Ile Arg Asn Glu Tyr Phe Glu Asn 195 200 205Asn Gln Leu Asn Leu Tyr Gln Met Arg Trp Asn Pro Glu Asn Pro Ala 210 215220 Gln His Gln Tyr Ala Ser Asp Ile Arg Trp Ala Asp Lys Ile Ala Lys 225230 235 240 Leu Met Asp Lys Ser Tyr Lys Gln Phe Gly Ile Lys Lys Asp AspIle 245 250 255 Arg Gln Thr Tyr Tyr Lys 260 786 base pairs nucleic aciddouble linear 3 GGTTTTGTCT ATATGAAGAA GAATTTCAAG TTACGCATTT CAACGCTACTATTGATAGTT 60 ATTTTAGTTG TTTTTGCTGT ATTACTCATC GTGAATGAAA CTAAATTGTTTAAAAATGAT 120 GTGAATTACT CTTTTGATGA GGCTGTTTCA ATGCAACAAG GGAAAGGTATTGTACAGACA 180 AAAGAAGAGG ATGGTAAATT TGTTGAAGCA AATAATAATG AAATTGCTAAAGCAATGACT 240 ATTTCACATA AAGACAATGA TATGAAGTAT ATGGATATCA CAGAAAAAGTGCCAATGTCG 300 GAATCTGAAG TTAACCAATT GCTAAAAGGT AAGGGGATTT TAGAAAATCGAGGGAAAGTT 360 TTTCTAGAAG CTCAAGAAAA ATATGAGGTT AATGTCATTT ATCTTGTTAGCCATGCATTA 420 GTAGAAACAG GTAACGGCAA ATCAGAATTA GCAAAAGGCA TTAAAGATGGGAAAAAACGC 480 TATTACAACT TTTTTGGTAT AGGAGCATTC GATAGTAGTG CTGTTCGTAGTGGGAAAAGT 540 TATGCTGAAA AGGAACAATG GACATCACCA GATAAGGCGA TTATTGGTGGTGCAAAGTTC 600 ATTCGTAATG AATATTTTGA AAACAATCAA CTGAATTTAT ATCAAATGCGATGGAATCCA 660 GAAAATCCTG CGCAACATCA ATATGCGAGT GACATTCGCT GGGCAGATAAAATTGCCAAA 720 TTAATGGATA AATCCTATAA GCAGTTTGGT ATAAAGAAAG ATGATATTAGACAAACATAT 780 TATAAA 786 262 amino acids amino acid single linear 4 GlyPhe Val Tyr Met Lys Lys Asn Phe Lys Leu Arg Ile Ser Thr Leu 1 5 10 15Leu Leu Ile Val Ile Leu Val Val Phe Ala Val Leu Leu Ile Val Asn 20 25 30Glu Thr Lys Leu Phe Lys Asn Asp Val Asn Tyr Ser Phe Asp Glu Ala 35 40 45Val Ser Met Gln Gln Gly Lys Gly Ile Val Gln Thr Lys Glu Glu Asp 50 55 60Gly Lys Phe Val Glu Ala Asn Asn Asn Glu Ile Ala Lys Ala Met Thr 65 70 7580 Ile Ser His Lys Asp Asn Asp Met Lys Tyr Met Asp Ile Thr Glu Lys 85 9095 Val Pro Met Ser Glu Ser Glu Val Asn Gln Leu Leu Lys Gly Lys Gly 100105 110 Ile Leu Glu Asn Arg Gly Lys Val Phe Leu Glu Ala Gln Glu Lys Tyr115 120 125 Glu Val Asn Val Ile Tyr Leu Val Ser His Ala Leu Val Glu ThrGly 130 135 140 Asn Gly Lys Ser Glu Leu Ala Lys Gly Ile Lys Asp Gly LysLys Arg 145 150 155 160 Tyr Tyr Asn Phe Phe Gly Ile Gly Ala Phe Asp SerSer Ala Val Arg 165 170 175 Ser Gly Lys Ser Tyr Ala Glu Lys Glu Gln TrpThr Ser Pro Asp Lys 180 185 190 Ala Ile Ile Gly Gly Ala Lys Phe Ile ArgAsn Glu Tyr Phe Glu Asn 195 200 205 Asn Gln Leu Asn Leu Tyr Gln Met ArgTrp Asn Pro Glu Asn Pro Ala 210 215 220 Gln His Gln Tyr Ala Ser Asp IleArg Trp Ala Asp Lys Ile Ala Lys 225 230 235 240 Leu Met Asp Lys Ser TyrLys Gln Phe Gly Ile Lys Lys Asp Asp Ile 245 250 255 Arg Gln Thr Tyr TyrLys 260 20 base pairs nucleic acid single linear 5 GTTAACCAAT TGCTAAAAGG20 21 base pairs nucleic acid single linear 6 AAACTGCTTA TAGGATTTAT C 21

What is claimed is:
 1. An isolated polynucleotide segment comprising anucleic acid sequence or the full complement of the entire length of thenucleic acid sequence, wherein the nucleic acid sequence is identical toSEQ ID NO:1 except that, over the entire length corresponding to SEQ IDNO:1, up to thirty nucleotides are substituted, deleted or inserted forevery 100 nucleotides of SEQ ID NO:1; wherein the nucleic acid sequenceis not genomic DNA and wherein the nucleic acid sequence detectsStaphylococcus aureus by hybridization.
 2. A vector comprising theisolated polynucleotide segment of claim
 1. 3. An isolated host cellcomprising the vector of claim
 2. 4. The isolated polynucleotide segmentof claim 1, wherein the nucleic acid sequence is identical to SEQ IDNO:1 except that, over the entire length corresponding to SEQ ID NO:1,up to ten nucleotides are substituted, deleted or inserted for every 100nucleotides of SEQ ID NO:1.
 5. The isolated polynucleotide segment ofclaim 1, wherein the nucleic acid sequence is identical to SEQ ID NO:1except that, over the entire length corresponding to SEQ ID NO:1, up tofive nucleotides are substituted, deleted or inserted for every 100nucleotides of SEQ ID NO:1.
 6. The isolated polynucleotide segment ofclaim 1, wherein the nucleic acid sequence is identical to SEQ ID NO:1except that, over the entire length corresponding to SEQ ID NO:1, up tothree nucleotides are substituted, deleted or inserted for every 100nucleotides of SEQ ID NO:1.
 7. An isolated polynucleotide segmentcomprising a nucleic acid sequence which hybridizes to the fullcomplement of SEQ ID NO:1, wherein the hybridization conditions includeincubation at 42° C. in a solution comprising: 50% formamide, 5× SSC(150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH7.6),5× Denhardt's solution, 10% dextran sulfate, and 20 micrograms/mldenatured, sheared salmon sperm DNA, followed by washing in 0.1× SSC at65° C.; wherein the nucleic acid sequence is identical to SEQ ID NO:1except that, over the entire length corresponding to SEQ ID NO:1, up tofive nucleotides are substituted, deleted or inserted for every 100nucleotides of SEQ ID NO:1; and, wherein the nucleic acid sequence isnot genomic DNA and wherein the nucleic acid sequence detectsStaphylococcus aureus by hybridization.
 8. The isolated polynucleotidesegment of claim 7, wherein the nucleic acid sequence is identical toSEQ ID NO:1 except that, over the entire length corresponding to SEQ IDNO:1, up to three nucleotides are substituted, deleted or inserted forevery 100 nucleotides of SEQ ID
 1. 9. A vector comprising the isolatedpolynucleotide segment of claim
 7. 10. An isolated host cell comprisingthe vector of claim
 9. 11. A vector comprising the isolatedpolynucleotide segment of claim
 8. 12. An isolated host cell comprisingthe vector of claim 11.